The silent information regulator 2 (Sir2) family of enzymes is conserved in organisms ranging from some bacteria to human. They have been implicated in many cellular processes such as the repair of DNA doublestrand breaks through nonhomologous end-joining, chromosome stability, aging, and gene silencing. Increased dosage of SIR2 genes promoted increased lifespan in yeast and C. elegans. The focus of this proposal is to determine the substrate specificity and the mechanism of catalysis by the Sir2 family of enzymes. Because these enzymes are present in many organisms and because they appear to be localized in different areas in the cell, it is possible that each has unique substrates. The authentic substrates for these enzymes have not been well established. The enzymatic function of Sir2 and its homologs, called sirtuins, were established just recently. It was demonstrated that sirtuins are NAD+-dependent deacetylases, catalyzing a unique reaction, which tightly couples NAD+ breakdown and substrate deacetylation to the formation of a novel compound, O-acetyl-ADP-ribose. We have recently shown that O-acetyl-ADP-ribose itself has a biological roles (unpublished data). How sirtuins carry out such a unique mechanism and produce a novel compound is still unknown. In this proposal, substrate specificity and the details of the catalytic mechanism will be determined.